Trazodone, dibenzoylmethane and tauroursodeoxycholic acid do not prevent motor dysfunction and neurodegeneration in Marinesco-Sjögren syndrome mice

Giada Lavigna; Anna Grasso; Chiara Pasini; Valentina Grande; Laura Mignogna; Elena Restelli; Antonio Masone; Claudia Fracasso; Jacopo Lucchetti; Marco Gobbi; Roberto Chiesa

doi:10.1371/journal.pone.0317404

Peer Review History

Original SubmissionJuly 22, 2024
22 Jul 2024 Author Response Attachments Attachment Submitted filename: 147177_1_rebuttal_3070929_sg099g.pdf https://doi.org/10.1371/journal.pone.0317404.r001
1 Dec 2024 Decision Letter - Shahbaz Ahmad Zakki, Editor PONE-D-24-30565Trazodone, dibenzoylmethane and tauroursodeoxycholic acid do not prevent motor dysfunction and neurodegeneration in Marinesco-Sjögren syndrome micePLOS ONE Dear Dr. Chiesa, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Jan 15 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Thank you for stating the following financial disclosure: “European Union:Roberto Chiesa PNRR-MR1-2022-12375730; Fondazione Telethon (FT):Roberto Chiesa GGP20071” Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 3. Thank you for stating the following in the Acknowledgments Section of your manuscript: “We thank David Ron for providing the CHO-KI cells stably transfected with the CHOP::luciferase reporter, and Angelini Pharma for providing trazodone. We are grateful to Stefano Fumagalli for advice on quantification of calbindin and CHOP immunostaining. This work was funded by Fondazione Telethon (GGP20071). Maintenance of the woozy mouse colony was partially supported by the European Union - Next Generation EU - NRRP M6C2 - Investment 2.1 Enhancement and strengthening of biomedical research in the NHS (PNRR-MR1-2022-12375730” We note that you have provided funding information that is currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: “European Union:Roberto Chiesa PNRR-MR1-2022-12375730; Fondazione Telethon (FT):Roberto Chiesa GGP20071” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 4. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The target article uses advanced methods to investigate potential treatments for Marinesco-Sjogren Syndrome (MSS). Despite testing several drugs and conducting extensive behavioral and histochemical analysis, the treatment failed to show disease modifying effects. This highlights the challenges of translating neuroprotective strategies. However, this study represents a significant step forward in understanding the limitations of current pharmacological approaches and points towards the exploration of alternative strategies for more effective interventions. Reviewer #2: General Comment: The manuscript provides a solid and methodologically sound investigation of the effects of trazodone, dibenzoylmethane (DBM), and tauroursodeoxycholic acid (TUDCA) on motor dysfunction and neurodegeneration in a preclinical mouse model of Marinesco-Sjögren syndrome (MSS). The study is comprehensive, well-executed, and the claims made are reasonable. However, there are some minor areas that require clarification and refinement to improve the manuscript's clarity and impact. Specific Comments: Comment #1: The abstract is detailed but could be made clearer or more focused. I suggest emphasizing the current primary findings, specifically the lack of neuroprotective effects, and discussing their potential implications for future MSS research if possible. This would provide readers with a clearer and more focused overview of the study’s outcomes. Comment #2: I suggest the author to provide more context for selecting trazodone, DBM, and TUDCA as the focus of this study. Expanding on the rationale behind testing these compounds specifically in MSS, as well as discussing the role of the PERK pathway in MSS pathophysiology, would strengthen the manuscript. It would also help to explain why these drugs were hypothesized to be effective in this context. Comment #3: While the manuscript includes a power analysis, it would benefit from a more detailed justification for selecting the 60% reduction in missteps as the target effect size. Clarifying this decision would improve the transparency and credibility of the study’s design. Additionally, further discussion on the rationale for the chosen dosing regimens particularly in relation to previous studies or pharmacokinetic data would provide a clearer foundation for the experimental approach. The manuscript could also clarify the relevance of the CHOP::luciferase assay to the in vivo findings, especially for readers who may be less familiar with molecular techniques. Finally, I encourage the authors to emphasize the translational challenges identified in the study and offer recommendations for future drug testing in MSS models. Comment #4: I recommend expanding the discussion to include a comparison of the findings with previous studies testing PERK inhibitors or chemical chaperones in other neurodegenerative models. Furthermore, addressing the lack of efficacy observed for the tested compounds and suggesting alternative approaches (e.g., targeting different pathways or exploring combination therapies) would be valuable. The pharmacokinetic findings could also benefit from further exploration. While the manuscript touches on the concentrations of TUDCA used in the CHOP::luciferase assays, a deeper explanation of how these concentrations compare to those used in vivo would help clarify whether the lack of effect observed in vivo could be due to non-physiological concentrations in vitro. Comment #5: The authors mention plans to explore trazodone’s mechanism of action further. I recommend expanding on this point by suggesting possible future studies that could overcome the limitations identified in this work. This might include exploring alternative models or approaches to better understand the drug’s effects in the context of MSS. Comment #6: The manuscript would benefit from a thorough review to address typographical and grammatical errors. Ensuring consistency in grammatical structure, particularly in the use of past and present tenses, and maintaining varied yet appropriate terminology would enhance readability. Avoiding repetitive use of the same words, such as "we," throughout the text would further improve the overall flow and clarity. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: Yes: Muhammad Ateeb ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. Attachments Attachment Submitted filename: Comment for Editor Manuscript PONE-D-24-30565.docx https://doi.org/10.1371/journal.pone.0317404.r002
Revision 1
18 Dec 2024 Author Response Reviewer #2 Specific Comments: Comment #1: The abstract is detailed but could be made clearer or more focused. I suggest emphasizing the current primary findings, specifically the lack of neuroprotective effects, and discussing their potential implications for future MSS research if possible. This would provide readers with a clearer and more focused overview of the study’s outcomes. We have revised the abstract to more clearly emphasize the lack of neuroprotective effect on Purkinje cell degeneration. We have also included a new sentence discussing the implication of our findings for future MSS research. Comment #2: I suggest the author to provide more context for selecting trazodone, DBM, and TUDCA as the focus of this study. Expanding on the rationale behind testing these compounds specifically in MSS, as well as discussing the role of the PERK pathway in MSS pathophysiology, would strengthen the manuscript. It would also help to explain why these drugs were hypothesized to be effective in this context. We have clarified the rationale for selecting trazodone, DBM, and TUDCA in the Introduction, referencing our perspective article published in Neural Regeneration Research (ref. 12), where we explain why these drugs were hypothesized to be effective in the context of MSS. Further details about the rationale for using trazodone and DBM are provided in the second paragraph of the Discussion, citing previous studies demonstrating their partial PERK inhibitory activity and neuroprotective effects in other neurodegenerative disease mouse models. We believe the current manuscript provides sufficient context for the study's objectives and would prefer not to add additional text to avoid redundancy or verbosity, unless the Editor advises otherwise. Comment #3: While the manuscript includes a power analysis, it would benefit from a more detailed justification for selecting the 60% reduction in missteps as the target effect size. Clarifying this decision would improve the transparency and credibility of the study’s design. Additionally, further discussion on the rationale for the chosen dosing regimens particularly in relation to previous studies or pharmacokinetic data would provide a clearer foundation for the experimental approach. The manuscript could also clarify the relevance of the CHOP::luciferase assay to the in vivo findings, especially for readers who may be less familiar with molecular techniques. Finally, I encourage the authors to emphasize the translational challenges identified in the study and offer recommendations for future drug testing in MSS models. We set a target effect size of a 60% reduction in missteps, as this approximates the effect previously observed when woozy mice were treated with the PERK inhibitor GSK2606414. This rationale has now been explicitly stated in the Study Design section of the Materials and Methods. Regarding the dosing regimens, these were chosen based on previously published studies in neurodegenerative disease models, as discussed in the manuscript. We believe this provides sufficient justification for the selected regimens. The CHOP::luciferase assay is a well-established method for assessing the effects of compounds on PERK pathway activation, and was included to validate the compounds’ activity at the molecular level. However, translating these results to the in vivo setting in terms of predictive efficacy and drug dosing is challenging, as we now state in the revised Discussion. We have emphasized the translational challenges identified in the study and offered alternative approaches (see points 1 and 4). Comment #4: I recommend expanding the discussion to include a comparison of the findings with previous studies testing PERK inhibitors or chemical chaperones in other neurodegenerative models. Furthermore, addressing the lack of efficacy observed for the tested compounds and suggesting alternative approaches (e.g., targeting different pathways or exploring combination therapies) would be valuable. The pharmacokinetic findings could also benefit from further exploration. While the manuscript touches on the concentrations of TUDCA used in the CHOP::luciferase assays, a deeper explanation of how these concentrations compare to those used in vivo would help clarify whether the lack of effect observed in vivo could be due to non-physiological concentrations in vitro. We believe the current manuscript provides a focused discussion of previous studies using PERK inhibitors and the chemical chaperone TUDCA in neurodegenerative disease mouse models. To maintain this focus, we prefer not to further expand the discussion to include additional chemical chaperones, as suggested by the reviewer. The manuscript already discusses alternative approaches, such as the use of a small-molecule Akt activator to enhance PERK phosphorylation at threonine 799 and the potential of gene therapy to restore SIL1 function. Given the limited understanding of other pathogenic mechanisms in MSS beyond maladaptive PERK-mediated UPR activation, discussing additional pharmacological strategies or combination therapies would be speculative and beyond the scope of this study. We agree with the reviewer that the >250 µM concentration of TUDCA active in the CHOP::luciferase assay is substantially higher than the submicromolar concentrations achieved in the brain of mice treated with the dosing regimens used in our study (0.4% in the diet or 500 mg/kg intraperitoneally). These dosing regimens were selected based on their demonstrated efficacy in previous neurodegenerative disease mouse models (refs 20, 21, 24-30). As explained in our response to Comment #3, the results obtained in vivo might not be directly related to the in vitro results in the CHOP::luciferase assay. Comment #5: The authors mention plans to explore trazodone’s mechanism of action further. I recommend expanding on this point by suggesting possible future studies that could overcome the limitations identified in this work. This might include exploring alternative models or approaches to better understand the drug’s effects in the context of MSS. In the manuscript, we speculate that the slight improvement in beam walking performance observed in trazodone-treated woozy mice may be related to its potential effects on serotonergic cerebellar transmission. To address the reviewer’s suggestion, we have added a brief sentence outlining how this hypothesis could be tested experimentally in future studies. Comment #6: The manuscript would benefit from a thorough review to address typographical and grammatical errors. Ensuring consistency in grammatical structure, particularly in the use of past and present tenses, and maintaining varied yet appropriate terminology would enhance readability. Avoiding repetitive use of the same words, such as "we," throughout the text would further improve the overall flow and clarity. The manuscript has been reviewed by a UK English editor with extensive experience in editing scientific papers. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0317404.r003
29 Dec 2024 Decision Letter - Shahbaz Ahmad Zakki, Editor Trazodone, dibenzoylmethane and tauroursodeoxycholic acid do not prevent motor dysfunction and neurodegeneration in Marinesco-Sjögren syndrome mice PONE-D-24-30565R1 Dear Dr. Roberto Chiesa, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Shahbaz Ahmad Zakki Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0317404.r004
Formally Accepted
3 Jan 2025 Acceptance Letter - Shahbaz Ahmad Zakki, Editor PONE-D-24-30565R1 PLOS ONE Dear Dr. Chiesa, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Shahbaz Ahmad Zakki Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0317404.r005

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